Rotation and flat-form imaging for microscopic objects

ABSTRACT

An example apparatus includes a well plate having an array of wells, a light encoding layer positioned under the well plate, an imaging layer to capture an image of the well plate encoded by the light encoding layer, an array of electrodes positioned on a surface of a bottom floor of the at least one well, and a controller. The light encoding layer is to encode light passing through a microscopic object in at least one well of the array of wells. The light encoding layer has a substantially flat form. The controller is to direct electrical voltage to the electrodes to generate a non-rotating, non-uniform electrical field, the electrical field being to rotate an object in the electrical field.

BACKGROUND

Analysis of biological material, such as cells, is performed for avariety of applications. Analysis is often performed by isolatingbiological material, such as cells or type of cells, and taking an imageof the biological material. The image may be taken using, for example, amicroscope. The image may then be analyzed for identification ordetection of various features of the biological material.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of various examples, reference is nowmade to the following description taken in connection with theaccompanying drawings in which:

FIG. 1 is a cross-sectional side view of an example apparatus forflat-form imaging;

FIG. 2 is a cross-sectional perspective view of another exampleapparatus for flat-form imaging;

FIG. 3 is a cross-sectional side view of the example apparatus of FIG. 1with an object to be imaged;

FIG. 4 is a cross-sectional side view of an example well with anelectrical field to rotate an object;

FIG. 5 is flow chart illustrating an example three-dimensional volumemodeling method;

FIG. 6 is a flow chart of another example method to generate athree-dimensional model of an object from images of the object;

FIG. 7 is a flow chart of another example method to generate athree-dimensional model of an object from images of the object;

FIG. 8 is a schematic illustration of an example system for flat-formimaging;

FIG. 9 is a schematic illustration of an example system for flat-formimaging; and

FIG. 10 is a flow chart illustrating an example method for flat-formimaging.

DETAILED DESCRIPTION

As noted above, analysis of biological material often includes imagingof the biological material. Such imaging often results in imaging of asingle sample or a small number of samples. Thus, monitoring ofreactions, for example, may be limited to a few samples at any one time,resulting in long sample-to-result times. Further, imaging systems areoften large due to the desired amplification of the samples. Forexample, a microscope with a long focal length may be employed to imagea microscopic sample.

In various examples, cells in a well plate with multiple wells may beimaged or monitored with a flat-form imaging arrangement. In oneexample, the well plate is provided with a light encoding layer and animaging layer. The light imaging layer may be a lens-less layer toprovide computational imaging (e.g., an amplitude mask or a diffuser) ora flat-lens array (e.g., meta-lens array). The light encoding layer mayencode the light from a light source passing through the cells in thewell plate. The raw imaging data may be captured by the imaging layer,which may include a CCD or a CMOS layer. The use of a flat-form lightencoding layer provides a large field of view for the imaging layer,allowing simultaneous imaging or monitoring of a well plate with a largenumber of wells. Each well is provided with an array of electrodes togenerate a non-uniform, non-rotating electric field. The electric fieldcreates an electrical torque to cause microscopic objects in the wellsto rotate. Thus, the microscopic object may be imaged from multipledirections.

Referring now to the Figures, a cross-sectional side view of an exampleapparatus 100 for flat-form imaging is illustrated. The exampleapparatus includes a well plate 110 which includes an array of wells120. In various examples, the well plate 110 may include any number ofwells 120. In the example illustrated in FIG. 1 , the array of wells 120includes a single well. In other examples, the number of wells 120 maybe as large as practically possible. In various examples, the well 120may have any of a variety of shapes. In the example of FIG. 1 , the well120 is defined by a side wall 122 and a bottom floor 124. The bottomfloor 124 has an internal surface 126 defining the interior of the well120.

The size and shape of each well 120 may be selected from any of avariety of sizes and shapes. In one example, the wells 120 arecylindrical with a cross section that may be circular, rectangular,hexagonal or any of a variety of other shapes. In other examples, eachwell 120 is conical or other non-cylindrical shape. In one example, eachwell 120 is a circular cylinder with a diameter of between 1 mm and 100mm.

The wells 120 are provided to position microscopic objects (not shown)therein. The well plate 110 may be formed of any of a variety ofmaterials such as, for example, plastics or glasses. In one example, thewell plate 110 is formed of a non-reactive material, such aspolypropylene. The well plate 110 may be of any practical size accordingto a desired application. For example, the well plate 110 may be sizedto accommodate a specific number of wells 120, each of which may beprovided to position the microscopic objects therein. The microscopicobjects may include any of a variety of objects such as cells orbiological reagents, for example.

The example apparatus 100 of FIG. 1 is provided with a light encodinglayer 130. The light encoding layer 130 of the example apparatus 100 hasa substantially flat form. For example, the light encoding layer 130 mayinclude a flat-form, lens-less computational imaging layer which mayinclude an amplitude mask or a diffuser, as described below withreference to FIG. 8 , or a substantially flat array of lenses (e.g.,meta-lens array), as described below with reference to FIG. 9 . Theflat-form nature of the light encoding layer 130 facilitates a widefield of view which can allow encoding of light over a large areasimultaneously. In this regard, light passing through a large number ofmicroscopic objects on a relatively large area of the well plate 110 canbe simultaneously encoded.

The example apparatus 100 of FIG. 1 further includes an imaging layer140 to capture an image encoded by the light encoding layer 130. Asnoted above, the image encoded by the light encoding layer 130 is thecollection of light passing through the well plate 110 and anymicroscopic objects on the well plate 110. In this regard, the imaginglayer 140 is able to capture an image of a wide field of view, asencoded by the light encoding layer 130. In various examples, theimaging layer may include a charge-coupled device (CCD) or acomplementary metal-oxide semiconductor (CMOS) device, for example.Thus, the example apparatus 100 is able to image a wide field of viewwith a flat form.

The example apparatus 100 of FIG. 1 is further provided with an array ofelectrodes 150 positioned on the surface 126 of the bottom floor 124 ofthe well 120. A controller 160 is provided to direct an electricalvoltage to the electrodes 150 from a power source (not shown in FIG. 1). In various examples, the power source is an alternating current (AC)power source. The electrical voltage causes the electrodes 150 togenerate an electrical field 170 in the well 120. In various examples,the electrical field 170 is a non-rotating, non-uniform electricalfield. The non-rotating, non-uniform electrical field 170 is provided torotate an object in the electrical field, as described below in greaterdetail with reference to FIG. 4 .

Referring now to FIG. 2 , a cross-sectional perspective view of anotherexample apparatus 200 is illustrated. The example apparatus 200 issimilar to the example apparatus 100 of FIG. 1 and includes a well plate210 with at least one well 220. The example apparatus 200 of FIG. 2further includes a light encoding layer 230 and an imaging layer 240,similar to the layers 130, 140 described above with reference to FIG. 1. As noted above, the well 220 may have any of a variety of shapes. Inthe example apparatus 200 of FIG. 2 , the well 220 is formed as arectangular prism with a rectangular bottom surface 222.

The example apparatus 200 is provided with an array of electrodes 250 onthe bottom surface 222 of the well 220. As described above, a controller260 is provided to direct an electrical voltage to the electrodes 250from an AC power source. The array of electrodes 250 may be positionedin a variety of manners. In certain examples, the electrodes 250 arearranged in a concentric formation. For example, in the exampleapparatus 200 of FIG. 2 , the electrodes 250 are rectangular electrodesarranged concentrically to one another. In other examples, theelectrodes may be arranged as concentric circles, concentric hexagons,or the like. The concentric electrodes are arranged to generate anelectrical field that is non-rotating and non-uniform.

Referring now to FIG. 3 , a cross-sectional side view of the exampleapparatus of FIG. 1 is illustrated with an object to be imaged. As notedabove, the example apparatus 100 of FIG. 1 is provided with an array ofelectrodes 150 to generate an electrical field 170 in the well 120. Invarious examples, the electrical field 170 is a non-rotating,non-uniform electrical field and is provided to rotate an object in theelectrical field. As illustrated in FIG. 3 , an object 310 (e.g., cell)may be positioned within the electrical field 170. The arrangement ofthe electrodes 150 allows the electrical field 170 to be non-rotatingwhile allowing the electrical field 170 to cause rotation of the object310 within the electrical field 170, as indicated by the arrow 312 inFIG. 3 and as described below with reference to FIG. 4 .

FIG. 4 is a cross-sectional side view of an example well with anelectrical field to rotate an object. FIG. 4 illustrates a well 400 withelectrodes 410 positioned on a bottom surface. As noted above, theelectrodes 410 are provided to generate a non-rotating, non-uniformelectrical field 420.

In one example, the electrical field produces a traveling wavedielectrophoresis (DEP) force in at least two directions. Whileelectrophoresis moves particles with the particles themselveselectrically charged, DEP results in movement of the particles withoutthe need for charges on the particles. Further, while stationary DEPrelies on an electrical field from adjacent electrodes having AC voltageat different frequencies, traveling wave DEP may achieve movement ofparticles with AC voltage applied to electrodes that are spaced apart(e.g., parallel or concentric) with a phase offset. In some examples,phase offset cycles through a 360-degree shift over a number ofelectrodes, such as four electrodes with adjacent electrodes having a90-degree phase offset. For example, the traveling wave DEP force may begenerated by applying voltage to the electrodes with phase variations.The phase variations result in the force in a direction based on thedirection of the phase offset. Thus, the particle travels in thedirection of the phase variation. In other words, the particle travelswhere the wave generated by the offset travels. As described in variousexamples above, the electrodes may be arranged in a concentric manner.

In one example, the voltage applied to the electrodes 410 may betemporally constant. Thus, the electrical field in the well is generatedby a temporally constant AC profile applied to the various electrodes410. Of course, those skilled in the art will appreciate that analternating current varies within a cycle. In this regard, a “constant”AC voltage refers to a voltage with a constant peak-to-peak voltage.

Referring again to FIG. 4 , the non-uniform nature of the electricalfield can result in an electrical torque applied to an object 430 in theelectrical field 420 when the object 430 is near an electrode (e.g.,electrodes 410) so as to rotate the object about a rotational axis 450parallel to the edge of the electrode. As indicated by the length of thefield lines in FIG. 4 , one portion of the object 430 may experience arelatively strong electrical field (e.g., the bottom portion of theobject 430), while another portion of the objet 430 may experience arelatively weak electrical field (e.g., the top portion of the object430). Thus, the non-uniform electrical field 420 can result in a torqueon the object 430, causing the object to rotate, as indicated by thearrow 440 about the axis 450. In various examples, the orientation ofthe electrical field may be controlled such that the axis of rotation450 can be aligned in different positions to selectively rotate theobject. In this manner, the object 430 can be imaged from any desireddirection.

The rotation of the object 430 facilitates the capturing of images ofthe object 430 from different angles to facilitate three-dimensionalreconstruction or modeling of cellular object 430 for analysis.Capturing of 2D images at various angles can allow for a transformationof the pixels at various angles into 3D voxels representing the object430. One example method of such transformation is described below withreference to FIG. 5 .

FIG. 5 is a flow chart of an example three-dimensional volumetricmodeling method 500. The example method 500 of FIG. 5 is described belowwith reference to the example system 100 described above with referenceto FIG. 3 . In this regard, the example method 500 may be carried out bya controller, such as the controller 160 of FIG. 3 , using capturedtwo-dimensional images of the rotating object 310. As indicated by block504, the controller 160 receives video frames or two-dimensional imagescaptured by the imaging layer 140 during rotation of object 310. Asindicated by block 508, various pre-processing actions are taken withrespect to the received two-dimensional images. Such preprocessing mayinclude filtering, binarization, edge detection, circle fitting and thelike.

As indicated by block 514, utilizing such edge detection, circle fittingand the like, controller 160 retrieves and consults a predefinedthree-dimensional volumetric template of the object 310, to identifyvarious internal structures of the object are various internal points inthe object. The three-dimensional volumetric template may identify theshape, size and general expected position of internal structures whichmay then be matched to those of the two-dimensional images taken at thedifferent angles. For example, a single cell may have athree-dimensional volumetric template comprising a sphere having acentroid and a radius. The three-dimensional location of the centroidand radius are determined by analyzing multiple two-dimensional imagestaken at different angles.

Based upon a centroid and radius of the biological object or cell, thecontroller 160 may model in three-dimensional space the size andinternal depth/location of internal structures, such as the nucleus andorganelles. For example, with respect to cells, the controller 160 mayutilize a predefined template of a cell to identify the cell wall andthe nucleus. As indicated by block 518 of FIG. 5 , using a predefinedtemplate, the controller 160 additionally identifies regions or pointsof interest, such as organs or organelles of the cell. As indicated byblock 524, the controller 160 matches the centroid of the cell membrane,nucleus and organelles amongst or between the consecutive frames so asto estimate the relative movement (R, T) between the consecutive framesper block 528.

As indicated by block 534, based upon the estimated relative movementbetween consecutive frames, the controller 160 reconstructs the centroidcoordinates in three-dimensional space. As indicated by block 538, thecentroid three-dimensional coordinates reconstructed from every twoframes are merged and aligned. A single copy of the same organelle ispreserved. As indicated by block 542, the controller 160 outputs athree-dimensional volumetric parametric model of object 310.

Referring now to FIG. 6 , a flow chart illustrating another examplemethod 600 to generate a model of an object from images of the object isprovided. A processor may perform elements of the method 600. At block602, the method 600 may include capturing a plurality of images of arotating object using the flat-form encoding 130 and imaging 140 layersdescribed previously with reference to FIGS. 1 and 3 . The images may becaptured over time, and the object may rotate into differentorientations between images.

Block 604 may include removing a first portion of a model of therotating object based on a first contour of the rotating object in afirst image of the plurality of images. For example, the first portionto be removed may be determined based on the first contour. The firstportion may be a portion outside the first contour or a portion insidethe first contour. At block 606, the method 600 may include orientingthe model based on an amount of rotation of the rotating object betweencapture of the first image and capture of a second image of theplurality of images. Orienting the model may include rotating the model,determining a location of an image plane relative to the model,selecting a projection direction based on the amount of rotation of therotating object, or the like.

Block 608 may include removing a second portion of the model of therotating object based on a second contour of the rotating object in thesecond image. For example, the second portion to be removed may bedetermined based on the second contour. In some examples, the first andsecond portions to be removed may both be determined before either isactually removed from the model. In an example, the first portion may beremoved from the model prior to determination of the second portion tobe removed.

Referring now to FIG. 7 , a flow chart illustrating another examplemethod 700 to generate a model of an object from images of the object isprovided. A processor may perform elements of the method 700. At block702, the method 700 may include measuring a rate of rotation of arotating object. In an example, the object may be subjected torotational forces of various magnitudes, such as electric field inducedby signals with various voltages or frequencies. The rate of rotationmay be measured to determine how the choice of parameters related toapplication of the rotational force affect the rate of rotation (e.g.,to allow parameters to be chosen to produce a desired rate of rotation).In some examples, a set of parameters related to application of therotational force may be chosen, and the rate of rotation may be measuredand stored for use during later elements of the method 700.

Block 704 may include selecting a frame rate of an imaging device basedon a rate of rotation of a rotating object or selecting a magnitude of anonrotating, nonuniform electric field to cause a rate of rotationselected based on a frame rate of the imaging device. For example, basedon the rate of rotation, the frame rate may be selected to result in adesired amount of rotation of the rotating object between capturedimages. In some examples, the frame rate may be fixed or set to apredetermined value, and the magnitude of an electric field to rotatethe rotating object may be selected based on that frame rate. In someexamples, the electric field may be a nonrotating, nonuniform electricfield that causes rotation of the rotating object. The magnitude of theelectric field, such as the voltage applied to electrodes to induce theelectric field, a frequency of the applied voltage, or the like, may beselected based on a desired rate of rotation of the rotating object. Thedesired rate of rotation may be selected based on the frame rate, forexample, to produce a desired amount of rotation of the rotating objectbetween captured images. The relationship between frequency or voltageand rate of rotation may be determined from the measurements at block702, may be calculated based on assumptions about the rotating object,or the like. In an example, the frame rate or voltage or frequency ofthe electric field may be selected without regard to the rate ofrotation of the rotating object.

At block 706, the method 700 may include applying a fluid in which therotating object is suspended to a substrate. For example, the rotatingobject may be mixed with the fluid or may have been previously stored inthe fluid. The fluid may be applied to the substrate by pipetting,jetting, or the like. The rotating object may not be rotating initiallywhen it is suspended in the fluid or applied to the substrate. Block 708may include rotating the rotating object by producing a nonrotating,nonuniform electric field using a plurality of electrodes on thesubstrate. Producing the nonrotating, nonuniform electric field mayinclude applying an AC voltage to a plurality of electrodes in or on thesubstrate (e.g., electrodes beneath the fluid). The voltage or frequencyof the AC voltage may be selected at block 704, may be predetermined, orthe like. In some examples, measuring the rate of rotation at block 702may be performed after block 708, such as when the voltage or frequencyof the AC voltage are not selected to produce a predetermined or designrate of rotation.

At block 710, the method 700 may include designing the light encodinglayer to provide a desired magnification. The magnification may beselected so that the rotating object can be imaged with a depth of fieldthat is at least substantially a depth of the object. For example,higher magnifications may result in smaller depths of field, so thehighest magnification that results in an acceptable depth of field maybe used.

Block 712 may include capturing a plurality of images of the rotatingobject using the imaging device, which is optically coupled lightencoding layer. For the imaging device may be instructed to capture aplurality of images, may be triggered at periodic intervals to capturethe plurality of images, or the like. At block 714, the method 700 mayinclude removing a first portion of a model of the rotating object basedon a first contour of the rotating object in a first image of theplurality of images. For example, removing the first portion may includedetermining the first portion to be removed, or removing the determinedfirst portion. In an example, determining the first portion to beremoved may include determining a first voxel to be removed.

Block 716 may include determining the amount of rotation of the rotatingobject between capture of the first image and capture of a second imageof the plurality of images based on the rate of rotation. The rate ofrotation may have been measured at block 708, may have been calculated(e.g., based on characteristics of the object, characteristics of theforce or field causing rotation, the voltage or frequency applied to anelectrode to create an electric field, etc.), may have been set bychoosing parameters related to generation of a force or field causingrotation (e.g., the voltage or frequency applied to an electrode tocreate an electric field, a magnitude of a field or force causingrotation, etc.), or the like. The amount of rotation may be determinedbased on the rate of rotation and the timing of the first and secondimages (e.g., the frame rate, the time between capturing of the firstand second images, or the like).

Block 718 may include orienting the model based on the amount ofrotation of the rotating object between capture of the first image andcapture of the second image. Orienting the model may include rotatingthe model, determining a location of an image plane relative to themodel, selecting a projection direction based on the amount of rotationof the rotating object, or the like. For example, the model may berotated the same amount the object rotated, or the image plane orprojection direction may have rotated the same amount as the object butin an opposite direction when the model is stationary.

Block 720 may include removing a second portion of the model of therotating object based on a second contour of the rotating object in thesecond image. As with block 714, removing the second portion may includedetermining the second portion to be removed, and removing thedetermined second portion. Determining the second portion to be removedmay include determining a second voxel to be removed. In an example,both the first portion and the second portion may be determined beforeeither portion is removed. For example, each contour may be compared tothe model to determine which portions to remove. In some examples, thecomparisons of multiple contours to the model may be used to determinewhether a particular portion (e.g., a particular voxel) can be removed.In some examples, the first portion may be removed before the secondportion is determined. In some examples, blocks of the method 700 may berepeated to iteratively remove portions of the model.

Referring now to FIG. 8 , a schematic illustration of an example system800 for flat-form imaging is illustrated. The light encoding layer iseither an amplitude mask used for lens-less computational imaging or bya meta-lens array. In both cases the distance between the light encodinglayer and the imaging layer is between 10 microns and 1 mm. The distancebetween the electrode plane in the well plate and the light encodinglayer is between 10 microns and 1 mm. This setup allows for a field ofview as wide as the imaging sensor (several square millimeters). Theexample system 800 includes a well plate 810 which includes at least onewell 820. The well plate 810 may be formed of any of a variety ofmaterials such as, for example, plastics or glasses. In one example, thewell plate 810 is formed of a non-reactive material, such aspolypropylene. The well plate 810 may be of any practical size accordingto a desired application. For example, the well plate 810 may be sizedto accommodate a specific number of wells 820. The well plate size canotherwise be matched to the imaging sensor size, or it can be bigger andthe imaging device would be scanned across the well plate surface toimage it completely.

In various examples, the well plate 810 of the example system 800 may beprovided with any number of wells 820. The size and shape of each well820 may be selected from any of a variety of sizes and shapes. In oneexample, the well 820 is cylindrical with a cross section that may becircular, rectangular, hexagonal or any of a variety of other shapes. Inother examples, the well 820 is conical or other non-cylindrical shape.In one example, the well 820 is a circular cylinder with a diameter ofbetween 1 mm and 100 mm.

Each well 820 may be provided for positioning of microscopic objects(not shown in FIG. 8 ) therein. As noted above, the microscopic objectsmay include biological material such as cells, for example. The wells820 are formed to allow positioning of the microscopic objects at ornear a bottom surface of the well 820 to facilitate imaging of themicroscopic objects from beneath the well plate 810.

Each well 820 is provided with an array of electrodes 850 formed on thebottom surface of the well. As described above, in various examples, thearray of electrodes 850 may be arranged in a concentric manner. Theelectrodes 850 may be provided with a voltage from an AC power source(not shown in FIG. 8 ). The AC power source may be coupled to acontroller 860 which selectively provides voltage from the AC powersource to the electrodes 850 in each well 820 of the well plate 810. Asdescribed above, the voltage from the AC power source provided to theelectrodes generates a non-rotating, non-uniform electrical field 870.

The example system 800 of FIG. 8 is provided with a light encoding layer830 positioned below the well plate 810 and an imaging layer 840positioned below the light encoding layer 830. A light source 890 ispositioned above the well plate 810 to illuminate the well plate 810.Thus, the light source 890 is positioned on one side of the well plate810, and the light encoding layer 830 and the imaging layer 840 arepositioned on a second, opposite side of the well plate 810.

Thus, the light encoding layer 830 is positioned to encode light fromthe light source 890 passing through the array of wells 820, as well asany microscopic objects therein. As noted above, in various example, thelight encoding layer 830 is provided with a substantially flat form.

In one example, the flat-form light encoding layer 830 includes alens-less, amplitude mask arrangement 832, as illustrated in the exampleof FIG. 8 . The amplitude mask arrangement 832 is provided to encode thelight passing through the well plate 810 for computationalreconstruction of the encoded information. The amplitude maskarrangement 832 includes a substrate 834 to support a patterned opaquelayer 836 and an isolating layer 838. Light passing through the wellplate 810 is passed through the isolating layer 838 and onto thepatterned opaque layer 836.

As noted above, the amplitude mask arrangement 832 can facilitatecomputational imaging. In this regard, computational imaging usesconversion of the incident light to sensor measurements. Rather thanrepresenting an image, the sensor measurements can be coupled with anappropriate algorithm or function to reconstruct an image. The algorithmor function may be determined through a calibration process.

In various examples, the amplitude mask arrangement 832 may be directlycoupled to the well plate 810. For example, the amplitude maskarrangement 832 may form the bottom surface of the well plate 810. Inthis regard, the isolating layer 838 may serve to provide isolation(e.g., chemical isolation) between the microscopic objects and thepatterned opaque layer. In various examples, the isolating layer 838 maybe formed of or coated with a fluorosilane or fluorinated paralyne toallow the microscopic objects to be rotated to allow 3D imaging. Suchisolation may prevent the microscopic objects from being affected by,for example, metals in the patterned opaque layer which may be toxic tothe microscopic objects.

The pattern of regions on the patterned opaque layer 836 is atwo-dimensional separable pattern to encode the light in 2-dimensionalregions. Each region may be sized to provide a resolution in thecaptured image of between 3 microns and 100 microns. Thus, the lightencoded by the amplitude mask arrangement 632 may be captured by theimaging layer 840 in the form of an M×N matrix of pixels. In variousexamples, the imaging layer 840 may include a CCD layer or a CMOS layer,each pixel being between 1 microns and 10 microns wide. As noted above,the pattern allows for a computational conversion of the encoded lightto an image through an appropriate algorithm or function. Thus, theexact pattern formed on the patterned opaque layer 836 may be anyfeasible pattern.

In one example, the patterned opaque layer 836 is formed with a fusedsilica glass wafer with a thin film of chromium deposited thereon. Thechromium is etched to form a pattern. In various example, the pattern isformed to provide a desired feature size which may correspond to a pixelin the imaging layer 840.

In some examples, the amplitude mask arrangement 832 may be replacedwith a diffuser layer (not shown). The diffuser layer may include alayer of a material with a non-uniform optical density, such as a thinsheet of thermally cured polymer or any semi-transparent coating.Various examples of diffuser layers may be fabricated withcost-efficient methods, such as fiber deposition, spin-coating or thelike. Such methods can achieve the desired result without the use ofspecialized equipment such as for photolithography. The non-uniformdensity of the diffuser layer can allow encoding of light which can beprocessed to produce a reconstructed image using, for example, areconstruction matrix 862, as described below.

The image captured by the imaging layer 840 may be processed by acontroller 860 coupled to the imaging layer 840. In various examples,the controller 860 may include a processor to execute variousinstructions. The controller 860 may be implemented as hardware,software, firmware, or a combination thereof. In one example, thecontroller 860 is implemented as software stored on a non-transitorycomputer-readable medium and includes instructions that may beexecutable by a processor.

The processing of the image from the imaging layer 840 may includetranslating the raw image from the imaging layer 840 by a reconstructionmatrix 862. The reconstruction matrix 862 may be obtained throughcalibration of the light encoding layer 830, for example. Thus, an arrayof pixels from the imaging layer 840 representing the raw image may bemultiplied by the reconstruction matrix 862 to obtain an array of pixelsrepresenting a reconstructed image. Thus, the flat-form light encodinglayer 830 can facilitate imaging of a wide field of view simultaneously.

In the example system 800 of FIG. 8 , the controller 860 is coupled to adispenser 880. The dispenser 880 is provided to drop, or inject, objectssuch as cells into the various wells 820 of the well plate 810. Invarious examples, the dispenser 880 may be provided to drop a singlecell at a time. The controller 860 may be coupled to the dispenser 880to move the dispenser to a selected location corresponding to a selectedwell 820. In other examples, the system 800 may include a movable stage(not shown) supporting the well plate 810. In this regard, thecontroller 860 may coordinate movement of the movable stage and thetiming of dropping of the cell from the dispenser 880 to drop the cellinto a desired, or selected, well 820 in the well plate 810.

In some examples, the dispenser 880 may inject or drop additionalmaterial into the wells 820. For example, the dispenser 880 may be usedto add stimuli onto cells already in the wells 820 to facilitate areaction or other response that may be observed or imaged. In otherexamples, the dispenser 880 may add fluorescent dyes or other stains tofacilitate the imaging.

Thus, as noted above, the example system 800 may be used to facilitateimaging of a wide field of view simultaneously. The wide field of viewmay include a large number of wells 820 of the well plate 810, forexample, thus allowing various types of analyses.

Referring now to FIG. 9 , a schematic illustration of another examplesystem 900 for flat-form imaging is illustrated. The example system 900of FIG. 9 is similar to the example system 800 described above withreference to FIG. 8 and includes a well plate 910 with various wells 920to position microscopic objects, a light encoding layer 930, an imaginglayer 940, a controller 960, a dispenser 980 and a light source 990.

Each well 920 is provided with an array of electrodes 950 formed on thebottom surface of the well. As described above, in various examples, thearray of electrodes 950 may be arranged in a concentric manner. Theelectrodes 950 may be provided with a voltage from an AC power source(not shown in FIG. 9 ). The AC power source may be coupled to acontroller 960 which selectively provides voltage from the AC powersource to the electrodes 950 in each well 920 of the well plate 910. Asdescribed above, the voltage from the AC power source provided to theelectrodes generates a non-rotating, non-uniform electrical field 970.

In the example system 900 of FIG. 9 , the light encoding layer 930 ismeta-lens arrangement 932. The meta-lens arrangement 932 includes ameta-lens layer 934 formed with an array of substantially flat lenses.In various examples, the array of lenses may be formed in a grid patterncorresponding to a grid of pixels groups in an image. In one example,the lenses in the meta-lens layer 934 are circular lenses with adiameter of between about 5 microns and about 200 microns. The thicknessof each lens and the meta-lens layer 934 is between about 20 nm andabout 1 microns. In various examples, the meta-lens layer 934 isprovided with spacer layers 936, 938 to properly position the array ofmeta-lenses relative to the microscopic objects. For example, the spacerlayers 936, 938 may provide the desired working distance for themeta-lenses in the layer 934.

In one example, the flat lenses of the meta-lens layer 934 may be lenseswith a high numerical aperture in the visible wavelengths. In thisregard, the lenses may have diffractive properties which provide adesigned or desired focal length. In other examples, the flat lenses areformed as transmissive dielectric metalenses. Such metalenses may beformed with TiO₂ nanofins formed on a glass substrate, as described inKhorasaninejad, Mohammadreza et al. (Jun. 3, 2016) Metalenses at visiblewavelengths: Diffraction-limited focusing and subwavelength resolutionimaging. Science Magazine, Pages 1190-1194.

The meta-lens arrangement 932 may thus encode light from the lightsource 990 passing through the well plate 910. Each meta-lens in themeta-lens layer 934 may correspond to a group of pixels in an imagecaptured by the imaging layer 940. The controller 960 can assemble thecomplete image with image pixels from the imaging layer 940.

Thus, the light encoding layer 930 with the meta-lens layer 934 providesflat-form imaging with a wide field of view. As described above withrespect to FIG. 8 , the example system 900 may thus be used tosimultaneously monitor a large number of samples for reactions orculture growth, for example.

Referring now to FIG. 10 , a flow chart illustrates an example method1000 for flat-form imaging. The example method 1000 includes projectinglight through a well plate having microscopic objects positioned thereon(block 1010). As noted above, in various examples, the well plate mayinclude an array of wells.

The example method 1000 further includes directing electrical voltage toelectrodes positioned within the wells (block 1020). The electricalvoltage is to generate a non-rotating, non-uniform electrical field. Theelectrical field can rotate the microscopic object in the electricalfield, as described above in the example of FIG. 4 .

The example method 1000 further includes encoding light passing throughthe sampling layer with a substantially flat-form light encoding layer(block 1030). In various examples, the light encoding layer may includean amplitude mask arrangement, as described above with reference to FIG.8 , or a meta-lens arrangement, as described above with reference toFIG. 9 , for example. The flat-form encoding layer provides a largefield of view to facilitate encoding of light passing through a largenumber of samples, for example.

The example method 1000 further includes imaging the microscopic objectson the sampling layer using encoded light from the flat-form lightencoding layer (block 1040). As noted above, a CCD device or a CMOSdevice may be used to capture an image using the encoded light from theflat-form light encoding layer.

Thus, the example systems described above provide an efficient andcost-effective imaging of a large number of samples. The use offlat-form light encoding allows imaging within a large field of view,allowing the large number of samples to be simultaneously monitored.

The foregoing description of various examples has been presented forpurposes of illustration and description. The foregoing description isnot intended to be exhaustive or limiting to the examples disclosed, andmodifications and variations are possible in light of the aboveteachings or may be acquired from practice of various examples. Theexamples discussed herein were chosen and described in order to explainthe principles and the nature of various examples of the presentdisclosure and its practical application to enable one skilled in theart to utilize the present disclosure in various examples and withvarious modifications as are suited to the particular use contemplated.The features of the examples described herein may be combined in allpossible combinations of methods, apparatus, modules, systems, andcomputer program products.

It is also noted herein that while the above describes examples, thesedescriptions should not be viewed in a limiting sense. Rather, there areseveral variations and modifications which may be made without departingfrom the scope as defined in the appended claims.

What is claimed is:
 1. An apparatus, comprising: a well plate having anarray of wells; a light encoding layer positioned under the well plate,the light encoding layer to encode light passing through a microscopicobject in at least one well of the array of wells, wherein the lightencoding layer has a substantially flat form and is to provide a widefield of view to encompass microscopic objects in the well plate; animaging layer to capture an image of the well plate, the image beingencoded by the light encoding layer; an array of electrodes positionedon a surface of a bottom floor of the at least one well; and acontroller to direct electrical voltage to the electrodes to generate anon-rotating, non-uniform electrical field, the electrical field beingto rotate an object in the electrical field.
 2. The apparatus of claim1, wherein the electric field is to generate an electrical torque on theobject when the object is near an electrode so as to rotate the objectabout a rotational axis parallel to the edge of the electrode.
 3. Theapparatus of claim 1, wherein the rotational axis is normal to theimaging layer.
 4. The apparatus of claim 1, wherein the light encodinglayer includes a flat-form, lens-less layer to facilitate computationalimaging.
 5. The apparatus of claim 4, wherein the flat-form lens-lesslayer includes at least one of an amplitude mask or a diffuser.
 6. Theapparatus of claim 1, wherein the light encoding layer includes asubstantially flat array of lenses.
 7. The apparatus of claim 1, whereinthe imaging layer includes at least one of a charge-coupled device (CCD)or a complementary metal-oxide semiconductor (CMOS) device.
 8. A system,comprising: a well plate including an array of wells; an array ofelectrodes formed on a bottom surface of each well in the array ofwells; an alternating current (AC) power source coupled to eachelectrode in the array of electrodes; a light encoding layer positionedbelow the well plate, the light encoding layer to encode light passingthrough the array of wells, wherein the light encoding layer has asubstantially flat form and is to provide a wide field of view toencompass microscopic objects in the well plate; an imaging layer tocapture the encoded image from the light encoding layer; and acontroller to: direct electrical voltage to the electrodes to generate anon-rotating, non-uniform electrical field, the electrical field beingto rotate an object in the electrical field; cause the imaging layer tocapture an encoded image of the object in at least two rotatedpositions; and generate a reconstructed image based on the encodedimages.
 9. The system of claim 8, wherein the electric field is togenerate an electrical torque so as to rotate the object about arotational axis.
 10. The system of claim 8, further comprising: adispenser to dispense samples into the array of wells.
 11. The system ofclaim 8, wherein the controller is to generate the reconstructed imagevia application of at least one transformation matrix to the encodedimage.
 12. The system of claim 8, wherein the controller is to monitorgrowth of samples in the array of wells.
 13. A method, comprising:projecting light through a well plate, the well plate having wells toposition microscopic objects therein; directing electrical voltage toelectrodes positioned within the wells to generate a non-rotating,non-uniform electrical field, the electrical field being to rotate themicroscopic object in the electrical field; encoding the light passingthrough the well plate with a substantially flat-form light encodinglayer, wherein the substantially flat-form light encoding layer providesa wide field of view to encompass the microscopic objects in the wellplate; and imaging the microscopic objects on the well plate usingencoded light from the flat-form light encoding layer.
 14. The method ofclaim 13, wherein the substantially flat-form light encoding layer is alens-less layer to facilitate computational imaging.
 15. The apparatusof claim 1, wherein the array of electrodes is arranged in a concentricformation.
 16. The apparatus of claim 15, wherein the array ofelectrodes is arranged as rectangular electrodes.
 17. The system ofclaim 8, wherein the light encoding layer comprises an amplitude maskwith a patterned opaque layer.
 18. The system of claim 17, wherein theamplitude mask further comprises an isolating layer.
 19. The system ofclaim 8, wherein the light encoding layer comprises a diffuser with anon-uniform optical density.
 20. The system of claim 8, wherein thelight encoding layer comprises a flat array of meta-lenses.